Avidin and streptavidin are tetrameric proteins that, due to their strong interaction with biotin, have become widely useful as extremely versatile, general mediators in a broad variety of bioanalytical applications, including chromatography, cytochemistry, cell cytometry, diagnostics, immunoassays and biosensing, gene probes and drug delivery (Wilchek and Bayer, 1990). The reason for their popularity is based on the basic principle of the avidin-biotin technology, namely, that the interaction between avidin and biotin is not affected when biotin is covalently bound to macromolecules or to insoluble carriers (matrices). The biotin moiety can be recognized by the native avidin molecule or derivatized avidin, which contains desired reporter groups.
Avidin (from egg-white) and streptavidin (from Streptomyces avidinii) are two related proteins that bind biotin with similar dissociation constants of about 10−15M. In addition to the binding of biotin, many of their physical properties are quite similar. Both, for example, are constructed of four non-covalently attached identical subunits, each of which bears a single biotin-binding site. The subunit Mr values are also very similar. Moreover, several short stretches in the sequences of the two proteins are preserved, particularly two Trp-Lys stretches that occur at approximately similar positions.
Despite these similarities, several differences exist between the two proteins. Avidin is a disulphide-bridged glycoprotein containing two methionine residues, whereas streptavidin is not glycosylated and is devoid of sulphur-containing amino acid side chains. Another significant difference is in the tyrosine content. Avidin has only one tyrosine residue (Tyr-33), whereas streptavidin has six tyrosine residues at positions 22, 43, 54, 60, 83 and 96. Interestingly, the single tyrosine residue of avidin is located in a region which contains a sequence identical with that of one of the streptavidin tyrosine residues (Tyr-43 in the stretch Thr-Gly-Thr-Tyr).
Each avidin monomer binds one molecule of biotin. The unique feature of this binding, of course, is the strength and specificity of formation of the avidin-biotin complex. The resultant affinity constant, estimated at 1.6×1015 M−for avidin and 2.5×1013 M−1for streptavidin, is the highest known for a protein and an organic ligand. It is so strong that biotin cannot be released from the binding site, even when subjected to a variety of drastic conditions such as high concentrations of denaturing agents at room temperature, e.g., 6 M guanidinium hydrochloride, 3 M guanidinium thiocyanate, 8 M urea, 10% β-mercaptoethanol or 10% sodium dodecyl sulfate. Under combined treatment with guanidinium hydrochloride at low pH (1.5) or upon heating (>70° C.) in the presence of denaturing agents or detergents, the protein is denatured, and biotin is dislodged from the disrupted binding site.
Avidin recognizes biotin mainly at the ureido (urea-like) ring of the molecule. The interaction between the binding site of avidin with the sulfur-containing ring of the valeric acid side chain of the vitamin is of much lower strength. The relatively weak interaction between the carboxy-containing side chain of biotin and avidin means that the former can be modified chemically and attached to a wide variety of biologically active material; the biotin moiety of the resultant derivative or conjugate is still available for interaction with avidin. In turn, the avidin can be derivatized with many other molecules, notably “probes” or reporter groups of different types.
This is the crux of avidin-biotin technology (Wilchek and Bayer, 1990). Thus, a biologically active target molecule in an experimental system can be “labeled” with its biotinylated counterpart (a binder), and the product can then be subjected to interaction with avidin, either derivatized or conjugated with an appropriate probe.
The use of the egg-white avidin in the avidin-biotin technology is sometimes restricted due to the high basicity (pI 10.5) and presence of sugar moieties on the avidin molecule, which may lead to nonspecific or otherwise undesired reactions. In recent years, the bacterial protein, streptavidin, has largely replaced egg-white avidin for most applications in avidin-biotin technology. However, the problems with streptavidin High cost and biotin-independent cell binding) have prompted renewed interest in egg-white avidin as the standard for avidin-biotin technology. For this purpose, modified avidins exhibiting improved molecular characteristics both over the native protein (and previous derivatives thereof) as well as over streptavidin, have been prepared, such as N-acyl avidins, e.g., N-formyl, N-acetyl and N-succinyl avidins. These derivatives of avidin reduce the charge of the protein, but they are all prepared via covalent attachment to the available lysines of avidin, and the consequent blocking of the free amino groups hinders subsequent preparation of other types of conjugates (notably protein-protein conjugates such as avidin-labeled enzymes) which are often prepared by crosslinking via lysine residues using bifunctional reagents (e.g. glutaraldehyde).
A more useful and effective alternative to lysine modification is the modification via arginines. In this case, the pI of the protein is efficiently reduced and the lysines are still available for subsequent interaction. Two different derivatives of avidin which are modified in this manner are commercially available. One, ExtrAvidin®, can be obtained in various functionally derivatized or conjugated forms from Sigma Chemical Company (St. Louis, Mo.). A second , NeutraLite Avidin™ (a product of Belovo Chemicals, Bastogne, Belgium) is additionally modified and can be purchased in bulk quantities.
Although the reduction of the pI of egg-white avidin solves one of the problems, the presence of the oligosaccharide residue remains a serious source of nonspecific (biotin-independent) interaction which restricts its application. The return of egg-white avidin as the standard for avidin-biotin technology has been contingent upon the removal of its sugars. The possibilities for removing a sugar from a glycoprotein are quite limited; it is possible to do so either chemically or enzymatically. The chemical methods currently available, e.g., using HF or periodate oxidation, are either destructive or inefficient. The well known enzymatic method, which employs N-glycanase, is usually very expensive and not very effective for avidin when conventional methodology is used. Eventually, a viable procedure for deglycosylation was established and the resultant product was subsequently modified chemically via the arginines and is known under the trade mark NeutraLite Avidin™ (Belovo Chemicals).
In spite of all these improvements, one of the main problems in the several applications of the avidin-biotin technology is the lack of an appropriate labeled avidin to permit the follow up of the binding of avidin to biotinylated compounds.
In addition to its interaction with biotin, avidin is known to associate non-covalently also with 4′-hydroxyazobenzene-2-carboxylic acid at the same biotin-binding site of the protein, but with a lower affinity (˜10−5–10−6 M) (Green, 1965). This non-covalent association is accompanied by a change in color from yellow to red (350 nm to 500 nm), thus allowing determination of avidin and its free binding sites.
Derivatives of 4′-hydroxyazobenzene-2-carboxylic acid and conjugates thereof with oligo and macromolecular carriers (HABAylated molecules) are the subject of copending application of Applicants filed at the same date as the present application.